Methods for producing chiral chromones, chromanes, amino substituted chromanes and intermediates therefor

ABSTRACT

Disclosed are process steps and novel processes for producing chromane compositions enriched in at least one (2R or 2S) enantiomer, preferably chroman-2-yl carboxylic acid compounds and chroman-2-yl carboxylic acid esters which are intermediates for producing platelet aggregation inhibitors and/or are themselves potent platelet aggregation inhibitors. Further disclosed are enzymatic processes for resolving chiral intermediates or final products to provide desired enantiomers.

FIELD OF THE INVENTION

[0001] This invention relates to novel processes for producing chromanecompounds, preferably chroman-2-yl acetic acid compounds and aminosubstituted chroman-2-yl acetic acid esters which are intermediates forproducing platelet aggregation inhibitors and/or are themselves potentplatelet aggregation inhibitors. It further relates to processes forresolving chiral intermediates or final products to provide desiredenantiomers.

BACKGROUND OF THE INVENTION

[0002] One process for making chromanes from coumarin derivatives isdescribed in U.S. Pat. No. 5,731,324 at pages 101-103. The unprotectedamino derivative bicyclic compound is shown on page 147. However, thatprocess involves chromatography as a purification step, which does notscale well commercially.

SUMMARY OF THE INVENTION

[0003] In accordance with one preferred embodiment, there is provided aprocess for making a compound, or a salt thereof, having a generalformula:

[0004] wherein R is C₁-C₈ alkyl and n=0 to about 3. The method comprises(a) through (f) below:

[0005] (a) reacting phenol and beta-keto glutaric acid in H₂SO₄/Ethanolwith heat, followed by pouring the reaction mixture onto ice water,extracting into organic solvent and evaporating as follows:

[0006] (b) hydrogenating the chromenone product from (a) above toproduce the corresponding chromanone:

[0007] (c) nitrating the chromanone from (b) as follows:

[0008] (d) resolving the racemic mixture using a lipase enzyme, asfollows:

[0009] (e) hydrogenating the 4-carbon to remove the oxo group andconvert the nitro group to an acetamido group as follows:

[0010] (f) acidifying the product from (e) above to recover the aminefollowed by addition of concentrated HCl to produce the HCl salt asfollows:

[0011] In accordance with one preferred embodiment, there is provided aprocess for making a compound, or a salt thereof, having a generalformula:

[0012] wherein R is C₁-C₈ alkyl and n=0 to about 3. The method comprises(a) through (g) below:

[0013] (a) reacting 2-hydroxyacetophenone and diethyloxalate in thepresence of sodium ethoxide followed by addition of concentratedsulfuric acid to make the bicyclic ring system as follows:

[0014] (b) hydrogenating the chromen-4-one to form the chroman4-one asfollows:

[0015] (c) performing a chain extension by first making the free acid,followed by reacting with borane-methyl sulfide complex to form the2-hydroxymethyl derivative, followed by replacing the hydroxy group witha tosyl group and reacting the tosyl derivative with a cyanide salt toform a 2-cyano derivative, followed by acidifying the cyano derivativein concentrated acid and esterifying the 2-acid group as follows:

[0016] (d) nitrating the product from (c) above to form the 6-nitrogroup as follows:

[0017] (e) resolving the racemic mixture using a lipase enzyme, asfollows:

[0018] (f) hydrogenating the 4-carbon to remove the oxo group andconvert the nitro group to an acetamido group as follows:

[0019] (g) acidifying the product from (f) above to recover the aminefollowed by addition of concentrated HCl to produce the HCl salt asfollows:

[0020] In accordance with one preferred embodiment, there is provided aprocess for making a compound, or a salt thereof, having a generalformula:

[0021] wherein R is C₁-C₈ alkyl and n=0 to about 3. The method comprises(a) through (e) below:

[0022] (a) reacting nitrophenol and diethyl ester of maleic acid withmethane sulfonic acid under heating as follows:

[0023] (b) performing a chain extension by first making the free acid,followed by reacting with borane-methyl sulfide complex to form the2-hydroxymethyl derivative, followed by replacing the hydroxy group witha tosyl group and reacting the tosyl derivative with a cyanide salt toform a 2-cyano derivative, followed by acidifying the cyano derivativein concentrated acid and esterifying the 2-acid group as follows:

[0024] (c) resolving the racemic mixture using a lipase enzyme, asfollows:

[0025] (d) hydrogenating the 4-carbon to remove the oxo group andconvert the nitro group to an acetamido group as follows:

[0026] (e) acidifying the product from (d) above to recover the aminefollowed by addition of concentrated HCl to produce the HCl salt asfollows:

[0027] In accordance with one preferred embodiment, there is provided aprocess for making a compound, or a salt thereof, having a generalformula:

[0028] wherein R is C₁-C₈ alkyl and n=0 to about 3. The processcomprises (a) through (g) below:

[0029] (a) nitrating the chromen4-one at the 6-position as follows:

[0030] (b) reacting the product from (a) above with TBSOTf to form abenzopyrillium salt as follows:

[0031] (c) adding the ketene enol to the benzopyrillium salt from (b)above as follows:

[0032] (d) acidifying the product from (c) above to complete theaddition of the substituent at the 2-position on the6-nitro-4-oxochromane ring as follows:

[0033] (e) resolving the racemic mixture using a lipase enzyme, asfollows:

[0034] (f) hydrogenating the 4-carbon to remove the oxo group andconvert the nitro group to an acetamido group as follows:

[0035] (g) acidifying the product from (f above to recover the aminefollowed by addition of concentrated HCl to produce the HCl salt asfollows:

[0036] The compositions formed according to the above methods preferablycomprise about 75% to about 100% of a single (2R) or (2S) enantiomer of6-aminochroman-2-yl acetic acid or an ester thereof.

[0037] In preferred embodiments, the lipase is from Pseudomonas cepacia,is the PS 30 lipase, is stabilized by cross-linking with alpha ketoglutarate and the like, or is the stabilized PS 30 enzyme ChiroCLEC-PC.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] In view of the shortcomings of the known process mentioned above,there is a need for improved processes for producing compounds that areuseful as intermediates in processes for producing platelet aggregationinhibitors. There is a particular need for improved processes for makingcompounds having the phenyl ring of the benzopyrans substituted by anamino group or a protected amino group. Such intermediates are usefulfor coupling with a carbonyl group to produce a carboxamide link andresult in compounds that are useful platelet aggregation inhibitors orintermediates for forming platelet aggregation inhibitors. Also neededis a process to produce relatively inexpensively large quantities ofchromone intermediates that are useful for being resolved byconventional processes to produce benzopyran or chromane derivativeswherein the chiral center at the two position of the saturated pyranring portion of the bicyclic ring structure can be resolved into racemicmixtures (R/S) that are enriched with one of the R or S enantiomers orto produce substantially pure compositions of a single enantiomer (R orS enantiomer). Due to inherent losses of up to 50% or more of thestarting materials (assuming a 50/50 R/S racemate) during enantiomericresolution, there is a need for a process which is efficient enough tobe scaled to an industrial level for inexpensively producing largequantities of a desired intermediate compound or large quantities offinal chroman-2-yl acetic acid ester compounds that are useful in theanticoagulant field.

[0039] Accordingly, there continues to be a need for a process that isadaptable to commercially scaleable production of such chromanes. One ormore of the foregoing needs may be met using the processes describedherein and the compounds and intermediates made thereby.

[0040] The disclosure herein presents novel processes for producingchromane compounds, preferably chroman-2-yl acetic acid compounds andamino substituted chroman-2-yl acetic acid esters which areintermediates for producing therapeutic agents, or are themselvestherapeutic agents, for disease states in mammals that have disorderscaused by or impacted by platelet dependent narrowing of the bloodsupply.

[0041] In particular, disclosed are processes that utilizes a SimonisChromone cyclizing step with a phenol starting material and glutarateketone under acidic condition (P₂O₅, phosphorous oxychloride or H₂SO₄).Preferably, sulfuric acid and absolute ethanol are utilized to producethe acetic acid ethyl ester at the 2-position as follows:

[0042] wherein R is a substituent on the phenyl or benzene ring and R isa nitro group or an amino group (or a protected amino group such as abenzamido or acetamido group) or a group that can be converted to anitro or amino group (e.g., hydrogen or halogen). Preferably, the2-carboxylic acid group is esterified with an ethyl group and the Rgroup on the benzene or phenyl portion is hydrogen, halogen or a NO₂group. Most preferably, the R group is a hydrogen atom.

[0043] The double bond of the oxo-pyran portion of the bicyclic ring ispreferably reduced by hydrogenation processes such as hydrogen and Pd/C,or the like as follows:

[0044] to produce the chromone product from the chromenone compound. Thepresence of ethyl acetate, ethanol and the like as a solvent canminimize the formation of a hydroxyl group from the carbonyl group inthe ring. In any event, a standard reaction to covert a hydroxyl groupto a ketone (carbonyl) may be alternatively utilized if a hydroxyl groupis desired. The resulting compound can be substituted with a desired Rsubstituent in the phenyl ring, preferably with a 6-position NO₂ group,by reacting the chromone compound with a nitrating agent such as ametallic nitrate in a mineral acid. Preferably, potassium nitrate isutilized with sulfuric acid at a temperature of from 0° C. to roomtemperature, but any nitration procedure may be used. Due to steric andelectronic directing, a very high amount of the 6-position nitrocompound is obtained and the reaction may be monitored with HPLC, forexample, to determine completion. Work-up is done by crystallization,e.g., ethyl acetate or toluene, to favor a particular position isomer.

[0045] The nitrating reaction may be illustrated as follows:

[0046] to provide a racemic ethyl (6-nitro-4-oxochroman-2-yl)acetate asshown above. Also, as is clear from the above discussion, if R is anitro group prior to the hydrogenation step, an amino group on thephenyl ring results from the hydrogenation with the Pd/C, or the like.Since certain lipases may favor the hydrolysis of the acetic acid esterwhen the nitro group is present on the phenyl ring, it is preferred thatthe nitro group be added to the phenyl ring after the hydrogenation stepis completed. The 6-position racemates, including the preferred 6-nitrocompound, may be generically illustrated as follows:

[0047] wherein R is a nitro group, an amino group or a protected aminogroup, such as an acetamido or benzamido group. The individualenantiomers of the racemate can be resolved as set forth below.

[0048] Alternatively, to avoid a hydrogenation step prior to nitration,the racemate set forth above can be produced via a benzopyrilium salt bytreating a 6-nitro-4-oxo-2-chromene nucleus with TBSOTf, and subjectingthe resulting intermediate to a silyl enol ether hydrolysis. The6-nitro-chromone nucleus is available commercially or can be produced,for example, by nitrating the chromone (4-oxo-2-chromene) at roomtemperature (Aldrich Catalog Number 19922-2). The nitration step may beillustrated as follows:

[0049] to selectively produce the 6-nitro-chromone nucleus in a highyield, since the electronic nature of chromone nucleus favors theplacement of the nitro group in the 6 position on the ring. Abenzopyrilium salt is then produced from this chromone nucleus asfollows:

[0050] Further, the silyl ketene acetal reactant can be produced fromthe acetic acid ethyl ester and TBSOTf by using standard methods in thesilyl ketene acetal art and reacted with the benzopyrilium salt that isset forth above, which was obtained from the 6-nitrochromoneintermediate. Reaction of the salt and the silyl ketene acetal areillustrated as follows:

[0051] Acidifying the above compound with aqueous HCl, or the likeresults in the desired racemate, 6-nitro-4-oxo-chroman-2-yl-acetic acid(ethyl ester), which can be resolved in the same manner as the racemateproduced by the Simonis Chromone cyclization procedure, see below.

[0052] The overall yield from the benzopyrilium salt to the racemate asset forth in the above alternative to the Simonis Chromone cyclizationprocedure is quite good, and is usually from about 85-95% yield. Oneembodiment, which summarizes the above reaction steps as well asenzymatic resolution of the racemate, is set forth below in Scheme IV.One might note that while the chromone and 6-nitro-chromone compoundsdescribed above are standard items of commerce, routine methods exist inthe art for producing such starting materials. Other obvious variationsand permutations of the above benzopyrilium salt procedure will bereadily apparent to one of ordinary skill in the art in view of thediscussion herein and are considered to be within the scope of thedisclosure.

[0053] A chirally selective lipase such as the Altus, Inc. ChiroCLEC-PClipase, or the like, may be utilized to resolve the ethyl2-(6-nitro-4-chromanone)acetate racemate, regardless of whether it isproduced by the Simonis Chromone cyclization or via the abovebenzopyrilium salt process. In the case where the nitro compound isutilized 98.5 percent of the acid formed corresponds to one enantiomer.Crystallization, or other standard separation procedures, may beutilized to separate the acid from the ester and result in asubstantially pure or enriched composition of a single enantiomer.Depending upon the desired enantiomer, the crystals or the supernatantmay be chosen to be used for further processing.

[0054] The undesired isomer may be recycled by using a racemization stepfollowed by re-exposure of the resulting racemate to the lipase. Theformation of a racemate from a single enantiomer is accomplished byexposing the enantiomer to a basic alcoholic solution such as a sodiumor potassium ethanolate solution. Other procedures which open the ringat the ring oxygen of the chromone and then reclose it may also beutilized to produce a racemate from a single enantiomer. By repeatingthe resolution and racemate forming steps, a higher overall yield may beobtained. The racemate forming step may be illustrated as follows:

[0055] wherein, as illustrated, a catalytic amount of potassiumcarbonate or similar catalytic base in R¹OH (preferably EtOH) isutilized for 1-3 days at room temperature, saponified with aqueous 1NNaOH in R¹OH for 3 hours. After acidification with 1N HCl, the racemicacid may be crystallized out, washed, and re-esterified with an acidicethanol solution. The resulting ester racemate can then be recycled byexposure to the lipase to obtain a higher yield of the desired singleenantiomer with respect to the initial amount of racemate startingmaterial.

[0056] After the resolution of the enantiomers, the 4-oxo group can beremoved from the (R) or (S) enantiomer chromone compound and the R groupcan be converted to an amino group by a simple hydrogenation reaction.Preferably, hydrogen gas and a Pd/C catalyst, or the like, are used toproduce a substantially pure single enantiomer of a (2 R or S)chroman-2-yl)acetic acid ester having the phenyl portion substituted asindicated above, preferably in the 6-position. For example, glacialacetic acid and 30-60 psi of hydrogen at 40-80° C. in the presence of acatalyst such as palladium on carbon may be utilized in a hydrogenator.The reaction can be monitored with HPLC to determine the completion ofthe hydrogenation. Molecular sieves may optionally be used as well.

[0057] Such a hydrogenation reaction is exemplified as follows:

[0058] wherein, the amino group can be in the form of a mineral acidsalt by addition of an acidic alcohol solution to the compound. Further,if the hydrogenation reaction results in creation of the free acid, itcan be converted to the desired ester by treatment with an appropriateester-forming alcohol in sulfuric acid followed by exposure to a mineralacid alcohol solution to provide the mineral acid salt of the aminogroup.

[0059] Non-limiting Illustrative Scheme I, set forth below, comprisesthe process steps outlined directly below which may also include furtherinitial starting steps to produce the starting materials which arecommercially available or further processing steps which modify theamino group to comprise a desired functional group, such as groupsdescribed in the anti-coagulation field. Amino coupling reactions arewell-known in the art. Moreover, specific steps that are set forth inthe preferred embodiment reaction scheme below are described in theexamples. The reaction products are isolated and purified byconventional methods, typically by solvent extraction into a compatiblesolvent. Preferred solvents are lower alkane ethers and alcohols; ethylether and isopropyl alcohol are preferred for solvent extraction orrecrystallization procedures. Esters of carboxylic acid side groups maybe formed that permit selective separation of the R and S enantiomers byuse of hydrolysis with a lipase, solvent extraction orrecrystallization. The products may be further purified by columnchromatography or other appropriate methods.

[0060] Alternatively, a corresponding 6-nitro-4-oxo-chroman-2-ylcarboxylic acid compound can be produced and esterified by using a5-nitro-acetophenone starting material and the reaction shown in the J.Med. Chem, Vol. 15, No. 8 (1972) or by nitration of the compounddescribed in the referenced article (see Scheme II, below).Additionally, the 6-nitro-4-oxo-chroman-2-yl carboxylic acid ester canbe made by reacting nitrophenol and the diethyl ester of maleic acid,for example (see Scheme III, below).

[0061] For example, the carboxylic acid group of the desired enantiomercan be extended to an acetic acid group by reducing the carboxylic acidside group to form a methanol side chain followed by extending itslength via a potassium cyanate reaction and the like. After the sideextension reaction is completed, the racemate of the6-nitro-4oxo-chroman-2-yl carboxylic acid ester can be resolved by theabove process utilizing the Altus ChiroCLECPC enzyme (or any otheracceptable lipase). PS 30 and functionally similar enzymes can also beutilized, and then extended to an acetic acid ethyl ester side chainafter the resolution.

[0062] After resolution, the 4-oxo group is removed and the 6-nitrogroup is reduced to an amino group by a hydrogenation step. For example,a hydrogenator loaded with the compound in ethanol or acetic anhydridein the presence of 10% palladium on carbon at 40-60° C. and 40-60 psi ofhydrogen can be used for the reduction step, with or without thepresence of molecular sieves. The progress of the reaction can bemonitored via HPLC.

[0063] Additional non-limiting schemes are set forth below:

[0064] In other embodiments, the order of some of the reactions in theschemes may be changed, and additional steps of protecting,deprotecting, nitrating, hydrolyzing, esterifying, and the like may beadded to the schemes at various points. Such minor alterations arewithin the scope of the disclosure herein. Although the esters shown areprimarily ethyl esters, other esters may be made, either by use ofdifferent solvents and/or reagents in the initial formation reactions orby transesterification.

[0065] The starting materials used in the disclosed processes arecommercially available from chemical vendors such as Aldrich, Lancaster,TCI, Bachem Biosciences, and the like, or may be readily synthesized byknown procedures including those present in the chemical literature, ormay be made by using procedures such as indicated above.

[0066] Reactions are carried out in standard laboratory glassware andreaction vessels under reaction conditions of standard temperature andpressure, except where it is otherwise indicated, or where use ofnon-STP conditions for a procedure is known in the art. Some procedures,reactions, and/or workups which are well known in the art or which arereadily available in standard reference texts in the art, includingBeilstein and Fieser and Fieser, may not be presented herein owing totheir stature of being within the knowledge of one of ordinary skill.Further, the above procedures of the claimed invention processes may becarried out on a commercial scale by utilizing reactors and standardscale-up equipment available in the art for producing large amounts ofcompounds in the commercial environment. Such equipment and scale-upprocedures are known to the ordinary practitioner in the field ofcommercial chemical production.

[0067] During the synthesis of these compounds, amino or acid functionalgroups may be protected by blocking groups to prevent undesiredreactions with the amino group during certain procedures. Procedures forsuch protection and removal of protecting groups are routine and wellknown to the ordinary practitioner in this field.

[0068] Enantiomeric Resolution and Acid Salt Formation

[0069] When a reaction results in the production of racemic chroman-2-ylcarboxylic acids and esters, these racemates are preferably resolved toproduce a mixture enriched in one of the R or S enantiomers orcompletely resolved into a substantially pure composition of one of theenantiomers. Examples of processes for resolving the racemic mixturesare provided herein and/or are known to those skilled in the art.Additionally, processes for the formation of acid addition salts such asthe hydrochloride salt of the 6-position amino acid group on thechromane nucleus are known in the art. Other such salts are alsoenvisioned.

[0070] Uses of Compounds

[0071] As mentioned above, the compounds produced according to preferredembodiments find utility as intermediates for producing therapeuticagents or as therapeutic agents for disease states in mammals, includingthose which have disorders that are due to platelet dependent narrowingof the blood vessels, such as atherosclerosis and arteriosclerosis,acute myocardial infarction, chronic stable angina, unstable angina,transient ischemic attacks and strokes, peripheral vascular disease,arterial thrombosis, preeclampsia, embolism, restenosis followingangioplasty, carotid endarterectomy, anastomosis of vascular grafts, andetc. These conditions represent a variety of disorders thought to beinitiated by platelet activation on vessel walls.

[0072] Platelet adhesion and aggregation is believed to be an importantpart of thrombus formation. This activity is mediated by a number ofplatelet adhesive glycoproteins. The binding sites for fibrinogen,fibronectin and other clotting factors have been located on the plateletmembrane glycoprotein complex IIb/IIIa. When a platelet is activated byan agonist such as thrombin, the GPIIb/IIIa binding site becomesavailable to fibrinogen, eventually resulting in platelet aggregationand clot formation. Thus, intermediate compounds for producing compoundsthat effective in the inhibition of platelet aggregation and reductionof the incidence of clot formation are useful intermediate compounds.

[0073] The compounds produced according to preferred embodiments mayalso be used as intermediates to form compounds that may be administeredin combination or concert with other therapeutic or diagnostic agents.In certain preferred embodiments, the compounds produced by theintermediates according to the present invention may be co-administeredalong with other compounds typically prescribed for these conditionsaccording to generally accepted medical practice such as anticoagulantagents, thrombolytic agents, or other antithrombotics, includingplatelet aggregation inhibitors, tissue plasminogen activators,urokinase, prourokinase, streptokinase, heparin, aspirin, or warfarin.The compounds produced from the intermediates may act in a synergisticfashion to prevent reocclusion following a successful thrombolytictherapy and/or reduce the time to reperfusion. Such compounds may alsoallow for reduced doses of the thrombolytic agents to be used andtherefore minimize potential hemorrhagic side-effects. Such compoundscan be utilized in vivo, ordinarily in mammals such as primates, (e.g.humans), sheep, horses, cattle, pigs, dogs, cats, rats and mice, or invitro.

[0074] Coupling Reaction of the Hydrochloride Salt IntermediateCompounds

[0075] The above compounds produced according to preferred methods maybe isolated and further reacted to substitute a desired group for one ormore of the hydrogen atoms on the amino group by a coupling reaction.Particularly preferred is a coupling reaction of the amino group with anacyl halide compound. For example, compounds such as5-amidino-thiophen-2-yl carboxylic acid derivatives (or an acyl halidesuch as the acyl chloride) and 4-amidinobenzoyl chloride may be coupledto ethyl (2S)-(6-aminochroman-2-yl) acetate (or its hydrochloride salt)to form ethyl(2S)-[6-(5-amidino-2-thiophenoyl)amino-chroman-2-yl]acetate and ethyl(2S)-{6-[(4-amidinophenyl) carbonylamino]chroman-2-yl} acetate, or othersimilar compounds or their derivatives which are known plateletaggregation inhibitors. For examples of such platelet aggregationinhibitors, see U.S. Pat. No. 5,731,324. The ring portion of the aboveamidino-aroyl or amidino-heteroaroyl derivatives may be substituted bygroups such as methyl, ethyl, fluoro, iodo, bromo, chloro, methoxy,ethyoxy, and the like which results in compounds that are known plateletaggregation inhibitors. Standard coupling procedures may be utilized,but procedures utilizing reaction mixtures the compounds, in salt form,are suspended in solvents such as acetonitrile, toluene, or the like,are preferred.

[0076] The compound formed from the coupling reaction may be used aseither the salt or the free base, and may be readily interconvertedbetween the two forms by using procedures which include those known inthe art as well as reacting the compound with one or more molarequivalents of the desired acid or base in a solvent or solvent mixturein which the salt is insoluble, or in a solvent like water after whichthe solvent is removed by evaporation, distillation or freeze drying.Alternatively, the free acid or base form of the product may be passedover an ion exchange resin to form the desired salt, or one salt form ofthe product may be converted to another using the same general process.The free base or salts may be purified by various techniques such asrecrystallization in a lower alkanol such as methanol, ethanol,propanol, isopropanol and the like, for example, or a mixture thereof.In preferred embodiments, the compound is recovered as the hydrochloridesalt and the recrystallization solvent is a 90/10-10/90 mixture ofethanol and isopropanol. Non-toxic and physiologically compatible saltsare preferred, although other types of salts may also be used, such asin the processes of isolation and purification.

[0077] Compositions and Formulations

[0078] Diagnostic and therapeutic applications of the compounds formedby procedures disclosed herein, including the aforementioned couplingreactions, will typically utilize formulations wherein the compound, ora pharmaceutically acceptable salt, solvate, or prodrug, is combinedwith one or more adjuvants, excipients, solvents, or carriers. Theformulations may exist in forms including, but not limited to tablets,capsules or elixirs for oral administration; suppositories; sterilesolutions or suspensions for injectable or parenteral administration; orincorporated into shaped articles. Subjects in need of treatment(typically mammalian) using the compounds of this invention can beadministered dosages that will provide optimal efficacy. The dose andmethod of administration will vary from subject to subject and bedependent upon such factors as the type of mammal being treated, itssex, weight, diet, concurrent medication, overall clinical condition,the particular compounds employed, the specific use for which thesecompounds are employed, and other factors which those skilled in themedical arts will recognize.

[0079] Formulations are prepared for storage or administration by mixingthe compound, or a pharmaceutically acceptable salt, solvate or prodrugthereof, having a desired degree of purity with physiologicallyacceptable carriers, excipients, stabilizers etc., and may be providedin sustained release or timed release formulations. Acceptable carriersor diluents for therapeutic use are well known in the pharmaceuticalfield, and are described, for example, in Remington's PharmaceuticalSciences, Mack Publishing Co., (A. R. Gennaro edit. 1985). Suchmaterials are nontoxic to the recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,acetate and other organic acid salts, antioxidants such as ascorbicacid, low molecular weight (less than about ten residues) peptides suchas polyarginine, proteins, such as serum albumin, gelatin, orimmunoglobulins, hydrophilic polymers such as polyvinylpyrrolidinone,amino acids such as glycine, glutamic acid, aspartic acid, or arginine,monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose or dextrins, chelatingagents such as EDTA, sugar alcohols such as mannitol or sorbitol,counter ions such as sodium and/or nonionic surfactants such as Tween,Pluronics or polyethyleneglycol.

[0080] Dosage formulations to be used for parenteral administration arepreferably sterile. Sterility is readily accomplished by filtrationthrough sterile membranes such as 0.2 micron membranes, or by otherconventional methods known to those skilled in the art. Formulations arepreferably stored in lyophilized form or as an aqueous solution. The pHof such preparations are preferably between 3 and 11, more preferablyfrom 5 to 9 and most preferably from 7 to 8. It will be understood thatuse of certain of the foregoing excipients, carriers, or stabilizerswill result in the formation of cyclic polypeptide salts. While thepreferred route of administration is by injection, other methods ofadministration are also anticipated such as intravenously (bolus and/orinfusion), subcutaneously, intramuscularly, colonically, rectally,nasally or intraperitoneally, employing a variety of dosage forms suchas suppositories, implanted pellets or small cylinders, aerosols, oraldosage formulations and topical formulations such as ointments, dropsand dermal patches. The compounds are desirably incorporated into shapedarticles such as implants which may employ inert materials such asbiodegradable polymers or synthetic silicones, for example, Silastic,silicone rubber or other polymers commercially available.

[0081] The compounds may also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of lipids, such as cholesterol, stearylamine orphosphatidylcholines.

[0082] The compounds may also be delivered by the use of antibodies,antibody fragments, growth factors, hormones, or other targetingmoieties, to which the compound molecules are coupled. The compounds mayalso be coupled with suitable polymers as targetable drug carriers. Suchpolymers can include polyvinylpyrrolidone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the plateletaggregation inhibitors may be coupled to a class of biodegradablepolymers useful in achieving controlled release of a drug, for examplepolylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross linked or amphipathic block copolymers of hydrogels. Polymers andsemipermeable polymer matrices may be formed into shaped articles, suchas valves, stents, tubing, prostheses and the like.

[0083] Therapeutic compound liquid formulations generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable byhypodermic injection needle.

[0084] Therapeutically effective dosages may be determined by either invitro or in vivo methods. For each particular compound and formulation,individual determinations may be made to determine the optimal dosagerequired. The range of therapeutically effective dosages will naturallybe influenced by the route of administration, the therapeuticobjectives, and the condition of the patient. For injection byhypodermic needle, it may be assumed the dosage is delivered into thebody's fluids. For other routes of administration, the absorptionefficiency must be individually determined for each inhibitor by methodswell known in pharmacology. Accordingly, it may be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect. The determination ofeffective dosage levels, that is, the dosage levels necessary to achievethe desired result, will be within the ambit of one skilled in the art.Typically, applications of compound are commenced at lower dosagelevels, with dosage levels being increased until the desired effect isachieved.

[0085] A typical dosage might range from about 0.001 mg/kg to about 1000mg/kg, preferably from about 0.01 mg/kg to about 100 mg/kg, and morepreferably from about 0.10 mg/kg to about 20 mg/kg. Advantageously, thecompounds or formulations may be administered several times daily, in aonce daily dose, or in other dosage regimens.

[0086] Typically, about 0.5 to 500 mg of a compound or mixture ofcompounds, as the free acid or base form or as a pharmaceuticallyacceptable salt or prodrug derivative (including esters), is compoundedwith a physiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, dye, flavor etc., as called for by acceptedpharmaceutical practice. The amount of active ingredient in thesecompositions is such that a suitable dosage in the range indicated isobtained.

[0087] Typical adjuvants which may be incorporated into tablets,capsules and the like are a binder such as acacia, corn starch orgelatin, and excipient such as microcrystalline cellulose, adisintegrating agent like corn starch or alginic acid, a lubricant suchas magnesium stearate, a sweetening agent such as sucrose or lactose, ora flavoring agent. When a dosage form is a capsule, in addition to theabove materials it may also contain a liquid carrier such as water,saline, a fatty oil. Other materials of various types may be used ascoatings or as modifiers of the physical form of the dosage unit.Sterile compositions for injection can be formulated according toconventional pharmaceutical practice. For example, dissolution orsuspension of the active compound in a vehicle such as an oil or asynthetic fatty vehicle like ethyl oleate, or into a liposome may bedesired. Buffers, preservatives, antioxidants and the like can beincorporated according to accepted pharmaceutical practice.

[0088] The compounds and formulations may be used alone or incombination, or in combination with other therapeutic or diagnosticagents. In certain preferred embodiments, the compounds and/orformulations may be coadministered along with other compounds typicallyprescribed for these conditions according to generally accepted medicalpractice, such as anticoagulant agents, thrombolytic agents, or otherantithrombotics, including platelet aggregation inhibitors, tissueplasminogen activators, urokinase, prourokinase, streptokinase, heparin,aspirin, or warfarin. The compounds and formulations can be utilized invivo, ordinarily in mammals such as primates, such as humans, sheep,horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.

[0089] The compounds, selected and used as disclosed herein, arebelieved to be useful for preventing or treating a conditioncharacterized by undesired thrombosis, such as (a) the treatment orprevention of any thrombotically mediated acute coronary syndromeincluding myocardial infarction, unstable angina, refractory angina,occlusive coronary thrombus occurring post-thrombolytic therapy orpost-coronary angioplasty, (b) the treatment or prevention of anythrombotically mediated cerebrovascular syndrome including embolicstroke, thrombotic stroke or transient ischemic attacks, (c) thetreatment or prevention of any thrombotic syndrome occurring in thevenous system including deep venous thrombosis or pulmonary embolusoccurring either spontaneously or in the setting of malignancy, surgeryor trauma, (d) the treatment or prevention of any coagulopathy includingdisseminated intravascular coagulation (including the setting of septicshock or other infection, surgery, pregnancy, trauma or malignancy andwhether associated with multi-organ failure or not), thromboticthrombocytopenic purpura, thromboanginitis obliterans, or thromboticdisease associated with heparin induced thrombocytopenia, (e) thetreatment or prevention of thrombotic complications associated withextracorporeal circulation (e.g. renal dialysis, cardiopulmonary bypassor other oxygenation procedure, plasmapheresis), (f) the treatment orprevention of thrombotic complications associated with instrumentation(e.g. cardiac or other intravascular catheterization, intra-aorticballoon pump, coronary stent or cardiac valve), and (g) those involvedwith the fitting of prosthetic devices.

[0090] Without further description, it is believed that one of ordinaryskill in the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds disclosed hereinand practice the claimed methods. The following working examplestherefore, specifically point out preferred embodiments, and are not tobe construed as limiting in any way the remainder of the disclosure.

EXAMPLES Example 1

[0091] Production of 2-ethoxycarbonyl-4-oxo-4H-benzopyran (ethyl4-oxochromene-2-carboxylate).

[0092] In a mixture containing 180 g of toluene and 12.0 g of diethyloxalate was dissolved 30 g of 2-hydroxyacetophenone, to which 65.0 g ofa 20% solution of sodium ethylate in ethanol was added dropwise. Aftercompletion of the reaction 13 g of 98% sulfuric acid was subsequentlyadded, and the mixture was stirred at 60° C. for about 30 minutes. Then140 g of water was added, and the mixture was subjected to separation ofthe organic layer. The resultant organic layer was concentrated, afterwhich 55.0 g of hexane was added and the mixture was filtered below 10°C. which yields about 34.0 g of ethyl 4-oxochromene-2-carboxylate.(Approximately 95% yield).

Example 2

[0093] Production of ethyl 4-oxochromane-2-carboxylate

[0094] A hydrogenator was charged by adding 6 g of ethyl4-oxochromene-2-carboxylate, 3.5 mL of acetic anhydride, 1 g of 10%palladium on carbon, 4.0 g of dried 3A molecular sieves (powered), and30 mL of glacial acetic acid. After purging several times with nitrogen,the hydrogenator was purged several times with hydrogen. Whilemaintaining stirring the reaction mixture was pressurized to about 30psi of hydrogen, heated to about 60° C. and maintained under thoseconditions for about 10-12 hours. HPLC monitoring of the reaction wasused to determine when the reaction was essentially complete, e.g., whenthe area ratio by HPLC between the chromen-4-one and the chroman-4-onewas not more than 3%. The mixture was then cooled to room temperatureand filtered through a celite bed. The catalyst and sieves were washedwith 10 mL aliquots of glacial acetic acid and the washes were combinedwith the filtrate. The combined mixture was concentrated under milddistillation conditions to an oil, which was dissolved with ethylacetate and extracted with saturated NaHCO₃. After the extraction, theaqueous layer was washed with ethyl acetate and the mixture neutralizedto a low pH with concentrated HCl. The mixtures was extracted severaltimes with ethyl acetate, the extracts were combined, concentrated to asolid, washed with acetonitrile and then filtered. Upon drying, about3.5-4.0 grams of ethyl 4-oxo-chromane-2-carboxylate were obtained as asolid.

Example 3

[0095] Production of ethyl 6-nitro-4-oxochromane-2-carboxylate

[0096] To a sulfuric acid solution of the ethyl4-oxo-chromane-2-carboxylate of Example 2 at −10° C. (about 3 ml) wasadded potassium nitrate in sulfuric acid (about 1 ml), wherein the molarratio of the potassium nitrate starting material to the chromonecompound starting material was slightly in excess of 1:1. The reactionmixture was stirred at about 0° C. for 1 hour, the ice bath was removedand the reaction mixture was stirred at room temperature for 4-6 hours.The nitrated chromone compound forms a precipitate and the reaction wasmaintained at room temperature until monitoring of the solution withHPLC shows the solution to be essentially free of the chromone startingmaterial (less than 3%). The reaction mixture was poured into ice andthe precipitate was extracted with ethyl acetate. The ethyl acetatelayer was dried, filtered and evaporated to give a light yellow solid.The yield of ethyl 6-nitro-4-oxochromane-2-carboxylate was approximately80%.

Example 4

[0097] Production of ethyl 6-nitro-4-oxochromane-2-carboxylate

[0098] In a mixture containing 1 mole of 4-nitrophenol were added 2moles of diethyl diester of maleic acid and 1120 ml methane sulfonicacid. The mixture was then heated to 92° C. for 20 hours. The reactionwas cooled to 0° C., poured onto 2 liters of ice and 2 liters of waterand extracted with 3 times with 800 mls of diethyl ether. The organiclayers were combined, washed with 3×500 ml water, 4×500 ml 1N NaOH,2×500 ml water, and 500 ml brine, dried over magnesium sulfate andconcentrated under vacuum to yield about 50-60 g of crude yellow solid,which was ethyl 6-nitro-4-oxochromane-2-carboxylate. (Approximately45%-65% yield).

Example 5

[0099] In situ ester Production of ethyl6-nitro-4-oxochromane-2-carboxylate

[0100] In a mixture containing 1 mole of 4-nitrophenol was added 2 molesof maleic acid and 500 mL of 1N sulfuric acid. The mixture was thenheated to 92° C. for 10-20 hours and monitored by HPLC for completion ofthe reaction with respect to the nitrophenol starting material. Thereaction was cooled to 50° C. and 2 moles of ethanol were added. Thetemperature maintained between 40° C.-55° C. for 1-3 hours or until HPLCindicates that the esterification was complete. The reaction was cooledto 0° C., poured onto 2 liters of ice and 2 liters of water andextracted with 3 times with 800 mls of diethyl ether (or ethanol). Theorganic layers were combined, washed with 3×500 ml water, 4×500 ml 1NNaOH, 2×500 ml water, and 500 ml brine, dried over magnesium sulfate andconcentrated under vacuum to yield about 75 g of crude yellow solid,which was ethyl 6-nitro-4-oxochromane-2-carboxylate.(Approximately>60-75% yield).

Example 6

[0101] Production of ethyl 2-(4-oxo-chromen-2-yl) acetate

[0102] In a mixture containing 1 mole of phenol was added 1.5 moles ofbeta-keto glutaric acid, 500 mL of 1N sulfuric acid and 200 mL ofethanol. The mixture was then heated to 80° C. for 10-20 hours andmonitored by HPLC for completion of the reaction with respect to thephenol starting material. The reaction was cooled to 0° C. and pouredonto 2 liters of ice and 2 liters of water and extracted with 3 timeswith 800 mls of diethyl ether (or ethanol). The organic layers werecombined, washed with 3×500 ml water, 4×500 ml 1N NaOH, 2×500 ml water,and 500 ml brine, dried over magnesium sulfate and concentrated undervacuum to yield about 60-70 g of a crude yellow solid, which was ethyl2-(4-oxo-chromen-2-yl) acetate. (Approximately>55-65% yield).

Example 7

[0103] Production of ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetate

[0104] The crude mixture obtained in Example 6 was reduced using 10%palladium on carbon substantially as set forth in Example 2, above, andthen nitrated substantially as set forth in Example 3, above, to yield aracemate of ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetate.(Approximately >80% yield with respect to the amount of startingmaterial obtain from Example 6).

Example 8

[0105] Production of 6-nitro-chromen-4-one

[0106] One gram molecular weight of 4-oxo-[4H]-benzopyran (AldrichCatalog Number 19,922-2), i.e., chromen-4-one, was nitrated andrecovered using the procedures set forth in Example 3, above, to yieldabout 92 g of 6-nitro-chromen-4-one, approximately 60% yield withrespect to the starting material.

Example 9

[0107] Production of benzopyrilium salt of 6-nitro-chromen-4-one

[0108] The crude material of Example 8 was reacted with TBSOTf understandard salt-forming conditions to yield the benzopyrilium salt inabout 95% yield.

Example 10

[0109] Production of ethyl 2-(4-[TBS-enol]-6-nitro-chroman-2-yl) acetate

[0110] The benzopyrilium salt of Example 9 was reacted with a molarexcess of the ketene enol resulting from TBSOTf reaction with the ethylester of acetic acid, by adding the ketene enol dropwise to thebenzopyrilium salt dropwise to produce the product in a 90% yield basedupon the benzopyrilium salt starting material.

Example 11

[0111] Production of ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetate

[0112] The 4-[TBS-enol] of Example 10 was converted to the ketone byacidify the reaction mixture with HCl and allowing the reaction mixtureto come to room temperature while stirring for 4-6 hours and thereaction mixture was cooled over ice. The precipitate crystals wereseparated extracted with ethyl acetate. The ethyl acetate layer wasdried and filtered to yield a light yellow solid in about 85-90% yieldwith respect to the benzopyrilium salt starting material of Example 9.The light yellow solid was the racemate of ethyl2-(6-nitro-4-oxo-chroman-2-yl) acetate.

[0113] Example 12 which follows provides a specific example of enzymaticresolution. However, this process may be used for compounds havingdifferent acid chain lengths and different ester groups, and may also beused for different types of lipase enzymes.

Example 12

[0114] Enzymatic Resolution of ethyl 2-(6-nitro-4-oxo-chroman-2-yl)acetate racemate

[0115] Approximately 330 g of the ethyl 2-(6-nitro-4-oxo-chroman-2-yl)acetate racemate of Example 11 was placed in isopropyl alcohol and waterin the presence of 1 g of the lipase enzyme ChiroCLEC-PC (Altus, Inc.)under the conditions set forth in the ChiroCLEC-PC Information Bookletavailable from Altus for 24 hours. After separation of the free acidproduct from the ester substrate, approximately 160 gm of the2-(2S)-6-nitro-4-oxochroman-2-yl-acetic acid was obtained in 98.5%purity.

[0116] Examples 13 and 14, which follow, provide a specific example ofconversion of a 6-nitro-4-oxo-chroman-2-yl acid to a6-amino-chroman-2-yl acid ester salt. The procedures of these examplesmay be used for compounds having esters other than ethyl and acid groupsother than acetic. In addition, acids and their corresponding esters aswell as salts and their corresponding free bases may be interconvertedusing methods known to those skilled in the art.

Example 13

[0117] Production of 2-[(2S) 6-acetamido-chroman-2-yl] acetic acid

[0118] A hydrogenator was charged by adding 6 g of 2-[(2S)6-nitro-4-oxo-chroman-2-yl] acetic acid, 3.5 mL of acetic anhydride, 1 gof 10% palladium on carbon, 4.0 g of dried 3A molecular sieves(powered), and 30 mL of glacial acetic acid. After purging several timeswith nitrogen, the hydrogenator was purged several times with hydrogen.While maintaining stirring the reaction mixture was pressurized to about70 psi of hydrogen, heated to about 80° C. and maintained under thoseconditions for about 10-12 hours. The bomb was then cooled to about 50°C., evacuated of hydrogen and purged several times with nitrogen.Trifluroroacetic acid (3.5 mL) was added the bomb, the bomb wasresealed, was purged several times with hydrogen and was thenpressurized to 70 psi of hydrogen. The reaction mixture was stirred asit was heated to 80° C. and was maintained at 80° C. with stirring. HPLCmonitoring was used to determine when the reaction was essentiallycompleted (the area ratio by HPLC between the intermediate and productwas not more than 3%) and the mixture was cooled to room temperature.After filtering of the mixture through a celite bed, the catalyst andsieves were washed with 10 mL aliquots of glacial acetic acid and thewashes combined with the filtrate. The combined mixture was concentratedunder mild distillation conditions to an oil, which was dissolved withethyl acetate and extracted with saturated NaHCO₃. After the extraction,the aqueous layer was washed with ethyl acetate and the mixtureneutralized to a low pH with concentrated HCl. The mixtures wasextracted several times with ethyl acetate, the extracts were combined,concentrated to a solid, washed with acetonitrile and then filtered.Upon drying, about 3.5-4.0 grams of ethyl2-((2S)-6-acetamido-chroman-2-yl) acetate were obtained as a whitesolid.

Example 14

[0119] Production of hydrochloride salt of ethyl 2-((2S)6-amino-chroman-2-yl) acetate

[0120] A mixture of 1.5 g of the ethyl 2-((2S)-6-acetamido-chroman-2-yl)acetate of Example 13, above, in 25 mL of concentrated sulfuric acid wasstirred vigorously at room temperature for about 6 hours, 50 mL ofethanol was added and the mixture was allowed to sit overnight. Theprecipitate was recovered by filtration and rinsed with 50 mL aliquotsof ether and dried. Absolute ethanol (25 mL) and HCl (10 mL) were mixedwith the precipitate for 2 hours followed by addition of 10 mL ofconcentrated HCl. The precipitate was recovered and recrystallized twicein an ether/isopropanol solvent mixture. Yielded was about 1.4 g ofethyl 2-((2S)-6-amino-chroman-2-yl) acetate hydrochloride (about 85-90%yield).

Example 15

[0121] Production of ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetateracemate

[0122] Approximately 330 g of the ethyl2-((2R)-6-nitro-4-oxo-chroman-2-yl) acetate of Example 12 was separatedin 99.4% purity from the (2S) isomer. To the (2R) enantiomer ester wasadded 500 mL of ethanol and a catalytic amount of potassium ethanolate(<1 equivalent). The mixture was maintained at room temperature for 28hours with stirring. Saponification was performed by addition of aqueous1 N NaOH in ethanol for 3 hours. After acidification with 1N aqueoushydrochloric acid the 6-nitro-4-oxo-chroman-2-yl acetic acid racematewas recovered as a precipitate in approximately 80% yield (about 250 g)with respect to the (2R) enantiomer ester starting material.

Example 16

[0123] Production of ethyl 2-(6-nitro-4-oxochroman-2-yl) acetateracemate

[0124] About 2.5 g of the crude precipitate of Example 15 was recoveredand added to 40 mL of concentrated sulfuric acid with vigorous stirringat room temperature for about 6 hours, 75 mL of ethanol was added andthe mixture was allowed to sit overnight. The precipitate was recoveredby filtration and rinsed with 60 mL aliquots of ether and dried.Absolute ethanol (40 mL) and HCl (20 mL) were mixed with the precipitatefor 2 hours. The precipitate was recovered and recrystallized twice inan ether/isopropanol solvent mixture. About 2.4 g of the ethyl2-(6-nitro-4-oxo-chroman-2-yl) acetate racemate was recovered (about85-90% yield), which can be recycled to Example 12 for enzymaticresolution of the enantiomers and a higher overall recovery of the (2S)enantiomer.

[0125] Examples 17-19 describe a specific procedure for lengthening a2-carboxyl chain on a chroman-4-one by one carbon. The procedure may beadapted to lengthen the chain by more than one carbon, including twocarbons and three carbons, by the use of appropriate reagents.

Example 17

[0126] Production of 2-(hydroxymethyl)-chroman-4-one

[0127] The carboxylic acid group of the compound of Example 2 (ethyl4-oxochromane-2-carboxylate) was treated with a metallic hydroxide basein ethanol, and then the aqueous layer was treated with an acid to formthe free acid, which was washed with water and then dried to yield 3 gof 4-oxo-chromane-2-carboxylic acid.

[0128] The 3 g of 4-oxo-chromane-2-carboxylic acid in THF (65 mL) wasstirred was cooled to 0° C. and borane-methyl sulfide complex (2.5 mL ofM solution, 25 mmoles) was added dropwise for about 15 minutes. Thesolution was warmed to room temperature and then heated at reflux for 4hours. The solution was cooled to room temperature and 10% aqueoushydrochloric acid (20 mL) was added over 15 minutes and the solution wasstirred at room temperature for 2 hours. The mixture was concentrated toapproximately 25 mL. The solution was poured into ethyl acetate (50 mL)and washed with water (2×30 mL), saturated sodium bicarbonate (2×30 mL)and saturated ammonium chloride (2×30 mL). The organic layer wasseparated dried over anhydrous MgSO4, and then concentrated in vacuo toyield about 2.6 g of 2-(hydroxymethyl)-chroman-4-one (86.7% yield).

Example 18

[0129] Production of 2-cyanomethylchroman-4-one

[0130] To a solution of 2.0 g of 2-(hydroxymethyl)-chroman-4-one (fromExample 17) in 35 mL of CH₂Cl₂ and 1.5 mL of pyridine was added 2 gp-toluenesulfonylchloride. The mixture was stirred at 25° C. for 36hours, then diluted with 20 mL ether and washed with 10 mL. The organiclayer was dried over MgSO₄ and concentrated to give 3.4 grams of crudetosylate. To the crude tosylate in 20 mL of DMSO was added with stirring80 mg of powdered sodium cyanide and the mixture was heated to refluxfor 1.5 hours under an inert atmosphere. The cooled mixture was dilutedwith 50 mL of water and extracted with 6 100 mL portions of ether andthe ether extracts were dried over anhydrous MgSO4 and filtered. Thefiltrate was concentrated and the residue was recrystallized withether/isopropanol to yield 1.7 grams of2-cyanomethylchroman-4-one.(about 85% yield).

Example 19

[0131] Production of ethyl 2-(4-oxochroman-2-yl) acetate

[0132] A mixture of 1.5 g of 2-cyanomethylchroman-4-one (Example 20) in25 mL of concentrated hydrochloric acid was stirred vigorously at roomtemperature for about 6 hours, 50 mL of ethanol was added and themixture was allowed to sit overnight. The precipitate was recovered byfiltration and rinsed with 50 mL aliquots of ether and dried. Absoluteethanol (25 mL) and HCl (10 mL) were mixed with the precipitate for 2hours followed by addition of 10 mL of concentrated HCl. The precipitatewas recovered and recrystallized twice in an ether/isopropanol solventmixture. Yielded was about 1.3 g of ethyl 2-(4-oxo-chroman-2-yl) acetate(about 80% yield).

Example 20

[0133] Production of ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetate,racemate

[0134] The 1.3 g of the ethyl 2-(4-oxo-chroman-2-yl) acetate, racemate(from Example 19) was nitrated using generally the procedures describedin Example 3 to yield 1.04 g of ethyl 2-(6-nitro-4-oxo-chroman-2-yl)acetate (80% yield).

Example 21

[0135] Production of ethyl 2-[(2S) 6-nitro-4-oxo-chroman-2-yl] acetate

[0136] The racemate of Example 20 was resolved into the respectiveenantiomers using the procedures in Example 12 to separate out the (2S)enantiomer. Alternatively, the free acid was formed and anesterification was performed in the present of the lipase as describe inthe ChiroCLEC-PC Information Booklet from Altus for 24 hours and the(2R) enantiomer was obtained instead of the (2S) enantiomer. Eitherenantiomer was obtained in 98.5% purity.

Example 22

[0137] Production of ethyl 2-(6-nitro-4-oxo-chroman-2-yl) acetateracemate from an Enriched ethyl 2-[(2R>2S) 6-nitro-4-oxo-chroman-2-yl]acetate Composition

[0138] Approximately 330 g of the ethyl ester of2-[(2R>2S)-6-nitro-4-oxo-chroman-2-yl] acetic acid of Example 12 wasseparated in about 98 purity for the (2R) isomer from the (2S) isomer.To the (2R) enantiomer ester was added 500 mL of ethanol and a catalyticamount of potassium ethoxide (<1 equivalent). The mixture was maintainedat room temperature for 28 hours with stirring. Saponification wasperformed by addition of aqueous 1 N NaOH in ethanol for 3 hours. Afteracidification with 1N aqueous hydrochloric acid the racemic (2Sapproximately equal to 2R) 2-(6-nitro-4-oxo-chroman-2-yl) acetic acidwas recovered as a precipitate in approximately 80% yield (about 250 g)with respect to the (2R) enantiomer ester starting material. The ethyl2-(6-nitro-4-oxo-chroman-2-yl) acetate racemate was formed from the freeacid using the general esterification procedures as set forth in Example14, above, which can be recycled to Example 12 for enzymatic resolutionof the enantiomers and a higher overall recovery of the (2S) enantiomer.

[0139] The procedures in the above examples directed to resolution ofthe 6-nitro substituted ethyl ester of the acetic acid (2S or 2R)chromanyl enantiomer from the racemate, can readily be adapted toresolution of the acetamido derivatives of the same structures and tonitro position isomers, as well as homologs of the compounds. Forexample, the enzymatic resolution procedures of Example 12, may bereadily adapted to the acetamido derivatives

[0140] The procedures above may be altered to use different startingmaterials, such as those having different substituents at the 6-position(amino, protected amino, hydrogen, etc.) Additionally, other minormodifications may be done, such as substitution of other known reagents,other catalysts, etc. Furthermore, other alcohols may be used to makeother esters, or the esters may be hydrolyzed to provide the free acid.

[0141] In view of the above description it is believed that one ofordinary skill can practice the invention. The examples given above arenon-limiting in that one of ordinary skill in view of the above willreadily envision other obvious permutations and variations withoutdeparting from the principal concepts embodied therein. Suchpermutations and variations are also within the scope of the disclosure.

What is claimed is:
 1. A process for making a compound, or a saltthereof, having a general formula:

wherein R is C₁-C₈ alkyl and n=0 to about 3, comprising: (a) reactingphenol and beta-keto glutaric acid in H₂SO₄/Ethanol with heat, followedby pouring the reaction mixture onto ice water, extracting into organicsolvent and evaporating as follows:

(b) hydrogenating the chromenone product from (a) above to produce thecorresponding chromanone:

(c) nitrating the chromanone from (b) as follows:

(d) resolving the racemic mixture using a lipase enzyme, as follows:

(e) hydrogenating the 4-carbon to remove the oxo group and convert thenitro group to an acetamido group as follows:

(f) acidifying the product from (e) above to recover the amine followedby addition of concentrated HCl to produce the HCl salt as follows:


2. A process for making a compound, or a salt thereof, having a generalformula:

wherein R is C₁-C₈ alkyl and n=0 to about 3, comprising: (a) reacting2-hydroxyacetophenone and diethyloxalate in the presence of sodiumethoxide followed by addition of concentrated sulfuric acid to make thebicyclic ring system as follows:

(b) hydrogenating the chromen-4-one to form the chroman-4-one asfollows:

(c) performing a chain extension by first making the free acid, followedby reacting with borane-methyl sulfide complex to form the2-hydroxymethyl derivative, followed by replacing the hydroxy group witha tosyl group and reacting the tosyl derivative with a cyanide salt toform a 2-cyano derivative, followed by acidifying the cyano derivativein concentrated acid and esterifying the 2-acid group as follows:

(d) nitrating the product from (c) above to form the 6-nitro group asfollows:

(e) resolving the racemic mixture using a lipase enzyme, as follows:

(f) hydrogenating the 4-carbon to remove the oxo group and convert thenitro group to an acetamido group as follows:

(g) acidifying the product from (f) above to recover the amine followedby addition of concentrated HCl to produce the HCl salt as follows:


3. A process for making a compound, or a salt thereof, having a generalformula:

wherein R is C₁-C₈ alkyl and n=0 to about 3, comprising: (a) reactingnitrophenol and diethyl ester of maleic acid with methane sulfonic acidunder heating as follows:

(b) performing a chain extension by first making the free acid, followedby reacting with borane-methyl sulfide complex to form the2-hydroxymethyl derivative, followed by replacing the hydroxy group witha tosyl group and reacting the tosyl derivative with a cyanide salt toform a 2-cyano derivative, followed by acidifying the cyano derivativein concentrated acid and esterifying the 2-acid group as follows:

(c) resolving the racemic mixture using a lipase enzyme, as follows:

(d) hydrogenating the 4-carbon to remove the oxo group and convert thenitro group to an acetamido group as follows:

(e) acidifying the product from (d) above to recover the amine followedby addition of concentrated HCl to produce the HCl salt as follows:


4. A process for making a compound, or a salt thereof, having a generalformula:

wherein R is C₁-C₈ alkyl and n=0 to about 3, comprising: (a) nitratingthe chromen-4-one at the 6-position as follows:

(b) reacting the product from (a) above with TBSOTf to form abenzopyrillium salt as follows:

(c) adding the ketene enol to the benzopyrillium salt from (b) above asfollows:

(d) acidifying the product from (c) above to complete the addition ofthe substituent at the 2-position on the 6-nitro-oxochromane ring asfollows:

(e) resolving the racemic mixture using a lipase enzyme, as follows:

(f) hydrogenating the 4-carbon to remove the oxo group and convert thenitro group to an acetamido group as follows:

(g) acidifying the product from (f) above to recover the amine followedby addition of concentrated HCl to produce the HCl salt as follows:


5. A composition produced by a method in any one of claims 1 through 4,comprising about 75% to about 100% of a single (2R) or (2S) enantiomerof 6-aminochroman-2-yl acetic acid or an ester thereof.
 6. A processaccording to any one of claims 1 through 4 wherein the enzyme is alipase from Pseudomonas cepacia.
 7. A process according to claim 6wherein the lipase is the PS 30 lipase.
 8. A process according to anyone of claims 1 through 4, wherein the lipase is a lipase stabilized bycross-linking with alpha keto glutarate and the like.
 9. A processaccording to claim 8, wherein the enzyme is the stabilized PS 30 enzymeChiroCLEC-PC.